Streptococcus suis polypeptides and polybnucleotides encoding same and their use in vaccinal and diagnostic applications

ABSTRACT

The present invention relates to the field of Streptococcus. More specifically, the present invention relates to the identification of polypeptides and polynucleotide sequences encoding the same which are involved in the pathogenic mechanism of S. suis. The present invention also relates to the use of such polypeptides in compositions and methods for the prevention, the treatment and diagnosis of S. suis-associated diseases and infections caused by S. suis.

This is an application for reissue of U.S. Pat. No. 7,927,608, and is areissue divisional of application Ser. No. 13/866,780, which is also anapplication for reissue of U.S. Pat. No. 7,927,608, now reissue U.S.Pat. No. Re. 45,467.

FIELD OF THE INVENTION

The present invention relates to the field of Streptococcus. Morespecifically, the present invention relates to the identification ofpolypeptides and polynucleotide sequences encoding the same which areinvolved in the pathogenic mechanism of S. suis. The present inventionalso relates to the use of such polypeptides in compositions and methodsfor the prevention, the treatment and diagnosis of S. suis-associateddiseases and infections caused by S. suis.

SEQUENCE LISTING

In accordance with 37 CFR §1.52(e)(5), the present specification makesreference to a Sequence Listing (submitted electronically as a .txt filenamed “SeqListing.txt” on Jan. 31, 2011 Feb. 27, 2015). The .txt filewas generated on Jan. 20, 2011 and is 40 Jan. 13, 2014 and is 48 kb insize. The entire contents of the Sequence Listing are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

Streptococcus suis is an important swine pathogen that causes manypathological conditions such as arthritis, endocarditis, meningitis,pneumonia and septicemia (13, 14). It is also an important zoonoticagent for people in contact with contaminated pigs or their by-products,causing meningitis and endocarditis (1, 36). Thirty-three serotypes(types 1 to 31, 33 and 1/2) based on capsular antigens are currentlyknown (9-11, 15, 17, 31). Type 2 is considered the most virulent andprevalent type in diseased pigs. The mechanisms involved in thepathogenesis and virulence of S. suis are not completely understood (13)and attempts to control the infection are hampered by the lack ofeffective vaccines.

Several approaches have been made to develop vaccines for S. suis.However, little success was achieved because the protection was eitherserotype or strain dependent and results, in most instances, wereequivocal (16, 30). For example, some protection with killed whole cellsand live avirulent vaccines were reported, but this required repeatedimmunization and the protection against heterologous challenges was notdetermined (18, 38). Exposure of young pigs with live virulent strainsshowed a positive effect in reducing clinical signs characteristics ofS. suis infection, but not in central nervous sign and mortality (35).Since the S. suis capsule plays an important role in virulence, attemptshave been made to develop a vaccine based on capsular material. However,this vaccination was unsatisfactory because the capsular polysaccharideis poorly immunogenic (7). More recently, interest has shifted towardprotein antigens of S. suis as vaccine candidates. Subunit vaccinesusing suilysin (20), or MRP (muramidase-released protein) and EF(extracellular proteins factor) (39) have been shown to protect pigsfrom homologous and heterologous serotype 2 strains, but their use ishindered by the fact that a substantial number of the virulent strainsin some geographical regions do not express these proteins (8, 12, 29).Thus, identification of other antigenic factors, especially surfaceproteins, could contribute to the development of a subunit vaccine.

There is thus a need for the discovery and use of new targets for theprevention, the treatment and the diagnosis of S. suis-associateddiseases and infections caused by S. suis.

SUMMARY OF THE INVENTION

An object of the invention is to fullfill the above-mentioned need. Morespecifically, the object is achieved by providing an isolatedpolypeptide comprising at least 15 contiguous amino acids in theN-terminal region of the amino acid sequence set forth in SEQ ID NO: 1.

Another object of the invention also concerns an polynucleotide encodinga polypeptide as defined above.

The present invention is further concerned with an antibody whichspecifically binds to a polypeptide of the invention.

A further object of the invention is to provide a vector comprising thepolynucleotide as defined above.

Yet another object of the invention is to provide a composition forpreventing or treating Streptococcus suis-associated diseases orinfection caused by S. suis, comprising an acceptable carrier and atleast one of the following elements:

-   -   a polypeptide as defined above;    -   a polypeptide as defined above;    -   an antibody as defined above;    -   a vector as defined above.

Another object of the invention concerns a method for treating and/orpreventing a Streptococcus suis-associated disease or infection in ananimal, the method comprising the step of administering to the animal acomposition as defined above.

A further object concerns a method for detecting the presence or absenceof a Streptococcus suis strain in a sample, comprising the steps of:

-   -   a) contacting the sample with an antibody of the invention for a        time and under conditions sufficient to form an immune complex;        and    -   b) detecting the presence or absence of the immune complex        formed in a).

Another object of the invention concerns a method for detecting thepresence or absence of antibodies raised against a Streptococcus suisstrain in a sample, comprising the steps of:

-   -   a) contacting the sample with a polypeptide of the invention for        a time and under conditions sufficient to form an immune        complex; and    -   b) detecting the presence or absence of the immune complex        formed in a).

The present invention also provide in another object a diagnostic kitfor the detection of the presence or absence of antibodies indicative ofStreptococcus suis strain, comprising:

-   -   a polypeptide according to the invention;    -   a reagent to detect polypeptide-antibody immune complex;    -   a biological reference sample lacking antibodies that        immunologically bind with said peptide; and    -   a comparison sample comprising antibodies which can specifically        bind to said peptide;        wherein said polypeptide, reagent, biological reference sample,        and comparison sample are present in an amount sufficient to        perform said detection.

Yet another object is to provide a diagnostic kit for the detection ofthe presence or absence of antibodies indicative of Streptococcus suisstrain, comprising:

-   -   an antibody of the invention;    -   a reagent to detect polypeptide-antibody immune complex;    -   a biological reference sample polypeptides that immunologically        bind with said antibody; and    -   a comparison sample comprising polypeptides which can        specifically bind to said peptide;        wherein said antibody, reagent, biological reference sample, and        comparison sample are present in an amount sufficient to perform        said detection.

Further objects are to provide an isolated polypeptide comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO 11 or functional derivative thereof, and an isolatedpolynucleotide encoding said polypeptide, and their use in a compositionand/or a method for treating and/or preventing a Streptococcussuis-associated disease or infection in an animal.

BRIEF DESCRIPTION OF THE FIGURES

Unless specifically indicated to the contrary, the terms “SP1” and “Sao”are used interchangeably.

FIG. 1: Schematic representation and partial restriction map of apreferred polynucleotide of the invention, namely the DNA insert ofrecombinant plasmid pSS735. Numbers indicate the distance (in basepairs) from the 5′ end.

FIG. 2: Nucleotide sequence (SEQ ID NO: 520) and deduced amino acidsequence (SEQ ID NO: 119) of the gene encoding a preferred polypeptideof a first embodiment of the invention, namely the SP1 (or Sao) proteinof S. suis. The Shine-Dalgarno sequence is in italic letters andunderlined. The initiation codon, ATG, and the stop codon, TAA, areshown in bold type. The two hydrophobic segments at the both N- andC-terminal ends of SP1 are underlined. The vertical arrow indicates thecleavage site of potential signal peptidase. R1 to R10 indicate thebeginning of the repeating units. The potential cell wall-associatedregion is underlined with dash line. The LPVTG (SEQ ID NO: 10) membraneanchor motif is boxed, and the charged C-terminal tail is indicated.

FIG. 3: Amino acid sequence alignment of the region Lys³¹⁹ to Val⁶⁰¹ ofSP1 (SEQ ID NO: 18) with the AvrXa7 a virulence factor of Xanthomonasoryzae pv. oryzae (SEQ ID NO: 17). The vertical lines indicate positionswith identical residues. Double dots represent conserved substitutionsand single dots represent functional substitutions.

FIG. 4: Expression of MBP-SP1 fusion protein in E. coli XL1-Blue andpurification of the recombinant mature SP1. The Coomassie-stained gel(A) and Western blot analysis (B) of the corresponding samples probedwith convalescent swine serum show E. coli whole cell lysate before(lane 1) and after (lane 2) induction of IPTG, extract of cytoplasm(lane 3), affinity purified MBP-SP1 fusion protein (lane 4), SP1 and MBPcleaved by factor X (lane 5) and recombinant SP1 devoid of MBP purifiedusing ion-exchange chromatography (lane 6). The molecular masses ofstandard proteins are indicated on the left.

FIG. 5: Immunoelectron microscopy of S. suis (4500×). The surfacelocation of SP1 on S. suis is demonstrated using a monospecific SP1antiserum and a gold-conjugated secondary antibody (B). No labeling wasfound in the control bacterial cell (A). Bars, 200 nm.

FIG. 6: Antibody responses after vaccination with the SP1 in piglets.(A) Total SP1-specific IgG in sera was measured by ELISA, showing thatsingle injection of SP1 elicited a significant IgG response that wasobviously enhanced by the booster. (B) ELISA for serum IgG isotypes inSP1 immunized pigs showed that IgG1 levels were consistently higher thanIgG2 levels. The results are expressed as the means of absorbances andstandard errors. *: p≦0.05.

FIG. 7: SP1-specific total humoral IgG titres in mice immunized withQuil A and Quil A plus SP1.

FIG. 8: IgG subclasses in sera from mice immunized with recombinant SP1.

FIG. 9: Vaccination with recombinant SP1 protects mice against S. suischallenge infection.

FIG. 10: Vaccination with recombinant SP1 protects mice from S. suisdeath.

FIG. 11: Nucleotide sequence (SEQ ID NO: 8) of a preferred functionalpolynucleotide fragment of the invention, namely the SP1A gene fragmentand the deduced amino acid sequences (SEQ ID NO: 4).

FIG. 12: Schematic representation and partial restriction map of the 6.3kb insert of recombinant phage. Numbers indicate the distance (in basepairs) from the 5′ end.

FIG. 13: Nucleotide sequence (SEQ ID NO: 12) and deduced amino acidsequence (SEQ ID NO: 11) of the gene encoding a preferred polypeptide ofanother embodiment of the invention, namely the SP2 protein of S. suis.The positive charge cluster at N-terminal end of SP2 is underlined. Thepotential N-terminal signal sequence is underlined with dash line. TheLysM domain is boxed, and the arrows indicate the beginning of therepeating units.

FIG. 14: Distribution of SP2 gene in different S. suis serotypes. TheSP2 genes were amplified by PCR from 31 of the 33 S. suis serotypereference strains.

FIG. 15: Expression of Trx-His-SP2 fusion protein in E. coli andpurification of the recombinant mature SP2. The Coomassie-stained gelshows E. coli whole cell lysate after induction of IPTG, affinitypurified Trx-His-SP2 fusion protein, SP2 and Trx-His cleaved byenterokinase, separated mature SP2 and Trx-His tag by an anion-exchangechromatography. The molecular masses are indicated on the left.

FIG. 16: Immunogenic and IgG-binding activity of recombinant SP2. a)SP2-specific rabbit serum reacts with the cell preparation of S. suisS735. b) Recombinant SP2 reacts with the convalescent swine serum.Recombinant SP2 binds to human (c) and pig (d) IgG.

FIG. 17: Antibody response after vaccination with recombinant SP2 inmice. SP2-specific IgG in sera was measured by ELISA.

FIG. 18: Vaccination with recombinant SP2 alleviates clinical signs ofthe mice challenged with a virulent S. suis strain.

FIG. 19: Vaccination with recombinant SP2 protects mice from S. suisdeath.

FIG. 20: Body temperature of pigs vaccinated with the compositionaccording to a preferred embodiment of the invention, after challenge.

FIG. 21: Clinical disease of pigs vaccinated with the compositionaccording to a preferred embodiment of the invention, after challenge.

FIG. 22: Survival of pigs vaccinated with the composition according to apreferred embodiment of the invention, after challenge.

FIG. 23: Serum total IgG titers of pigs vaccinated with the compositionaccording to a preferred embodiment of the invention.

FIG. 24: IgG subclasses induced from pigs vaccinated with thecomposition according to a preferred embodiment of the invention.

FIG. 25: Amino acid sequence alignment between two SP1 polypeptidesaccording to preferred embodiments of the invention, namely SEQ ID NO 1and 2.

FIG. 26: Amino acid sequence alignment between two SP1 polypeptidesaccording to preferred embodiments of the invention, namely SEQ ID NO 1and 3.

FIG. 27: Amino acid sequence alignment between two SP1 polypeptidesaccording to preferred embodiments of the invention, namely SEQ ID NO 2and 3.

FIG. 28: Amino acid sequence alignment between three SP1 polypeptidesaccording to preferred embodiments of the invention, namely SEQ ID NO 1,2 and 3.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have surprisingly found two novel S. suis polypeptides andpolynucleotides encoding same that are involved during the S. suispathogenic mechanism. In this connection, the present inventionspecifically relates to their identification and to the use of saidpolypeptides or polynucleotides in compositions and methods for theprevention, the treatment and the diagnosis of Streptococcussuis-associated diseases or infection caused by S. suis.

A non-exhaustive list of Streptococcus suis-associated diseases whichthe methods of the invention may be useful for, includes those, such asarthritis, endocarditis, meningitis, pneumonia and septicemia.

Definitions

The term “isolated” is meant to describe a polynucleotide, a polypeptideor an antibody that is in an environment different from that in whichthe polynucleotide, the polypeptide, the antibody, or the host cellnaturally occurs.

The term “animal” refers to any animal susceptible to be infected by aStreptococcus strain, such as S. suis. Specifically, such an animal maybe, but not limited to, mice, pig, sheep, horse and human. Morespecifically, the animal consists of a pig.

The term “treating” refers to a process by which the symptoms of aninfection or a disease associated with a Streptococcus strain arealleviated or completely eliminated. As used herein, the term“preventing” refers to a process by which symptoms of an infection or adisease associated with a Streptococcus strain are obstructed ordelayed.

The term “protective response” means prevention of onset of aStreptococcus suis-associated disease or an infection caused by S. suis.or lessening the severity of such a disease existing in an animal. Thelevel of “protective response” may be evaluated, for instance, by theassignment of clinical scores such as those defined in Example 4.

The expression “an acceptable carrier” means a vehicle for containingthe compounds obtained by the method of the invention that can beadministered to an animal host without adverse effects. Suitablecarriers known in the art include, but are not limited to, goldparticles, sterile water, saline, glucose, dextrose, or bufferedsolutions. Carriers may include auxiliary agents including, but notlimited to, diluents, stabilizers (i.e., sugars and amino acids),preservatives, wetting agents, emulsifying agents, pH buffering agents,viscosity enhancing additives, colors and the like.

The term “fragment”, as used herein, refers to a polynucleotide sequence(e.g., cDNA) which is an isolated portion of the subject nucleic acidconstructed artificially (e.g., by chemical synthesis) or by cleaving anatural product into multiple pieces, using restriction endonucleases ormechanical shearing, or a portion of a nucleic acid synthesized by PCR,DNA polymerase or any other polymerizing technique well known in theart, or expressed in a host cell by recombinant nucleic acid technologywell known to one of skill in the art.

1 Polynucleotides and Polypeptides of the Invention

In a first embodiment, the present invention concerns an isolatedpolypeptide which consists of a surface protein, and more particularly aC-terminal-anchored surface protein of Streptococcus suis, namely calledSP1 or Sao (Genbank accession number AY864331). As shown in the Examplesection, the SP1 polypeptide advantageously elicits a protectiveresponse to a Streptococcus suis strain challenge when administered toan animal, such as a pig.

Specifically, the isolated polypeptide of the first embodiment of theinvention comprises at least 15 or even preferably at least 25 or evenmore at least 35 contiguous amino acids in the N-terminal region of theamino acid sequence set forth in SEQ ID NO: 1. As one skilled in the artmay appreciate, the term “N-terminal region” in the context of thepresent invention when referring to the Sao protein, preferably consistsof the region spanning from amino acid residue 1 to 293 of the aminoacid sequence set forth in SEQ ID NO: 1.

According to a preferred embodiment, the isolated polypeptide of thefirst embodiment may further comprises at least one repetitive aminoacid sequence such as shown in FIG. 2. More particularly, a repetitiveamino acid sequence contemplated by the present invention consists ofthe amino acid sequence shown in SEQ ID NO 9,

wherein

Xaa₁ is Val, Thr or Ile;

Xaa₃ is Lys or Glu;

Xaa₄ is Lys or Glu;

Xaa₅ is Ala or Gln;

Xaa₇ is Thr or Pro;

Xaa₈ is Gly, Ser or Val;

Xaa₉ is Lys, Val, Ile or Asn;

Xaa₁₀ is Glu or Val;

Xaa₁₁ is Lys or Asn;

Xaa₁₂ is Gly, Glu or Asp;

Xaa₁₃ is Asn or Met;

Xaa₁₄ is Ile, Ala or Val;

Xaa₁₅ is Glu or Val;

Xaa₁₆ is Pro or Thr;

Xaa₁₈ is Glu or Gln;

Xaa₁₉ is Lys or Glu;

Xaa₂₂ is Thr or Ala

Xaa₂₆ is Lys or Asn;

Xaa₂₇ is Asp or Glu;

Xaa₂₈ is Asn or Lys;

Xaa₂₉ is Ile or Val and

Xaa₃₀ is Glu or Val.

It will be understood that the preferred SP1 polypeptide of theinvention may comprises only one of said repetitive sequence, whereas insome other cases, the preferred SP1 polypeptide of the invention maycomprises at least two repetitive sequences or even more than ten ofsuch repetitive sequences.

In accordance with another preferred embodiment of the invention, theisolated SP1 polypeptide advantageously comprises at least 15 or evenpreferably 25 or even more preferably 35 contiguous amino acids in theC-terminus region of the amino acid sequence set forth in SEQ ID NO: 1.Preferably, the C-terminus region comprises a membrane anchor motif,such as the one consisting of the amino acid sequence as set forth isSEQ ID NO 10, namely Leu Pro Val Thr Gly.

As one skilled in the art may appreciate, the term “C-terminus region”in the context of the present invention when referring to the SP1protein, preferably consists of the region spanning from amino acidresidue 593 to 670 of the amino acid sequence set forth in SEQ ID NO: 1.

In accordance with an even more preferred embodiment, a SP1 polypeptideof the invention comprises an amino acid sequence substantiallyidentical to a sequence selected from the group consisting of SEQ ID NOS1 to 3 or functional derivative thereof. Most preferably, a SP1polypeptide of the invention consists of an amino acid sequencesubstantially identical to the sequence shown in SEQ ID NO 1, or afunctional derivative thereof.

A “functional derivative”, as is generally understood and used herein,refers to a protein/peptide sequence that possesses a functionalbiological activity that is substantially similar to the biologicalactivity of the whole protein/peptide sequence. In other words, itpreferably refers to a polypeptide or fragment(s) thereof thatsubstantially retain(s) the capacity of eliciting an immune response,such as a protective response to a S. suis strain challenge when saidfunctional derivative is administered to an animal. A preferredfunctional derivative contemplated by the present invention comprises anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO 4. More specifically, a preferred functional derivativeconsists of a 315-amino acids fragment (S²⁸-K³⁴²) of SEQ ID NO 1. Such afragment or polypeptide is designed as SP1A and its nucleotide and aminoacid sequences are shown in FIG. 11. SP1A strongly reacted with aconvalescent swine serum in immunoblots and immunization withrecombinant SP1A elicits significant humoral antibody responses in pigsand mice, demonstrating that SP1A is highly immunogenic. (See Example 5)

According to a second embodiment, the present invention relates toanother isolated S. suis polypeptide, namely called SP2, whichadvantageously elicits a protective response in an animal.

Specifically, the isolated polypeptide of the second embodiment of theinvention comprises an amino acid sequence substantially identical to asequence as set forth in SEQ ID NO: 11 or functional derivative thereof.

By “substantially identical” when referring to an amino acid sequence,it will be understood that the polypeptide of the present inventionpreferably has an amino acid sequence having at least 75% homology, oreven preferably 85% homology, or even more preferably 95% homology topart or all of the sequence shown in SEQ ID NOS 1 to 4 and 11.

“Homology” in this context, means identical or similar to the referencedsequence while straightforward replacements/modifications of any of theamino acids provided, are included as well. A homology search in thisrespect can be performed with the BLAST-P (Basic Local Alignment SearchTool), a program well known to those of skill in the art. For thecorresponding nucleic acid sequence, homology is referred to the BLASTXand BLASTN programs known in the art.

The present invention also concerns an isolated polynucleotide encodinga preferred SP1 or a preferred SP2 polypeptide of the invention.Preferably, the isolated polynucleotide of the invention comprises anucleotide sequence substantially identical to the sequence shown in SEQID NOS 5 to 7 when referring to SP1 and SEQ ID NO 12 when referring toSP2 and their respective functional fragments thereof.

By “substantially identical” when referring to a nucleic acid sequence,it will be understood that the polynucleotide of the inventionpreferably has a nucleic acid sequence which is at least 65% identical,more particularly 80% identical and even more particularly 95% identicalto part or all of the sequence shown in SEQ ID NOS 5 to 7 and 12 orfunctional fragments thereof.

A “functional fragment”, as is generally understood and used herein,refers to a nucleic acid sequence that encodes for a functionalbiological activity that is substantially similar to the biologicalactivity of the whole nucleic acid sequence. In other words, and withinthe context of the present invention, it preferably refers to a nucleicacid or fragment(s) thereof that substantially retains the capacity ofencoding a polypeptide/protein which elicits an immune response, andmore preferably a protective response, to a Streptococcus suis strainchallenge when administered to an animal. For instance, such a fragmentis the polynucleotide shown in SEQ ID NO 8, which codes for the SP1Apolypeptide as defined above.

In another embodiment, the invention is further directed to vector(e.g., cloning or expression vector) comprising a polynucleotide of theinvention as defined above.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector.

A number of vectors suitable for stable transfection of cells andbacteria are available to the public (e.g., plasmids, adenoviruses,baculoviruses, yeast baculoviruses, plant viruses, adeno-associatedviruses, retroviruses, Herpes Simplex Viruses, Alphaviruses,Lentiviruses), as are methods for constructing such cell lines. It willbe understood that the present invention encompasses any type of vectorcomprising any of the polynucleotide molecule of the invention.

2. Antibodies

In another embodiment, the invention features antibodies thatspecifically bind to the polypeptides of the invention. Morespecifically, the antibody is a purified polyclonal or monoclonalantibody that specifically binds to the preferred S. suis polypeptidesas defined above.

The antibodies of the invention may be prepared by a variety of methodsknown to one skilled in the art. For example, the polypeptides of theinvention may be administered to an animal in order to induce theproduction of polyclonal antibodies. Alternatively, and as mentionedabove, antibodies used as described herein may be monoclonal antibodies,which are prepared using known hybridoma technologies (see, e.g.,Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., 1981; Charland, N., M. Jacques, S. Iacouture and M.Gottschalk. 1997. Characterization and protective activity of amonoclonal antibody against a capsular epitope shared by Streptococcussuis serotypes 1, 2 and 1/2. Microbiology 143 (Pt 11): 3607-14).

With respect to antibodies of the present invention, the term“specifically binds to” refers to antibodies that bind with a relativelyhigh affinity to one or more epitopes of the SP1 or SP2 polypeptide ofthe invention, but which do not substantially recognize and bindmolecules other than the SP1 or SP2 polypeptides of the invention. Asused herein, the term “relatively high affinity” means a bindingaffinity between the antibody and the SP1 or SP2 polypeptides of atleast 10⁶ M⁻¹, and preferably of at least about 10⁷ M⁻¹ and even morepreferably 10⁸ M⁻¹ to 10¹⁰ M⁻¹. Determination of such affinity ispreferably conducted under standard competitive binding immunoassayconditions which are common knowledge to one skilled in the art.

3. Methods of Treatment and Compositions

The SP1 and SP2 polypeptides, polynucleotides encoding same andantibodies of the invention may be used in many ways in the treatmentand/or prevention of Streptococcus suis-associated diseases or infectioncaused by S. suis.

For instance, and according to an aspect of the invention, the SP1and/or SP2 polypeptides of the invention may be used as immunogens forthe production of specific antibodies for the treatment and/orprevention of Streptococcus suis infection. Suitable antibodies may bedetermined using appropriate screening methods, for example by measuringthe ability of a particular antibody to passively protect againstStreptococcus suis infection in a test model. Examples of an animalmodel are the mouse and pig models described in the examples herein.

According to another aspect, the polynucleotides encoding polypeptidesof the invention or derivatives thereof may be used in a DNAimmunization method. That is, they can be incorporated into a vectorwhich is replicable and expressible upon injection thereby producing theantigenic polypeptide in vivo. For example polynucleotides may beincorporated into a plasmid vector under the control of the CMV promoterwhich is functional in eukaryotic cells. Preferably the vector isinjected intramuscularly.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method or system such asdirect injection of plasmid DNA into muscles [Wolf et al. H M G (1992)1: 363, Turnes et al., Vaccine (1999), 17: 2089, Le et al., Vaccine(2000) 18: 1893, Alves et al., Vaccine (2001) 19: 788], injection ofplasmid DNA with or without adjuvants [Ulmer et al., Vaccine (1999) 18:18, MacLaughlin et al., J. Control Release (1998) 56: 259, Hartikka etal., Gene Ther. (2000) 7:1171-82, Benvenisty and Reshef, PNAS USA (1986)83: 9551, Singh et al., PNAS USA (2000) 97: 811], targeting cells bydelivery of DNA complexed with specific carriers [Wa et al., J Biol Chem(1989) 264: 16985, Chaplin et al., Infect. Immun. (1999) 67:6434],injection of plasmid complexed or encapsulated in various forms ofliposomes [Ishii et al., AIDS Research and Human Retroviruses (1997) 13:142, Perrie et al., Vaccine (2001) 19:3301], administration of DNA withdifferent methods of bombardment [Tang et al., Nature (1992) 356: 152,Eisenbraun et al., DNA Cell Biol (1993) 12: 791, Chen et al., Vaccine(2001) 19:2908], and administration of DNA with lived vectors [Tubulekaset al., Gene (1997) 190: 191, Pushko et al., Virology (1997) 239: 389,Spreng et al. FEMS (2000) 27: 299, Dietrich et al., Vaccine (2001) 19:2506].

A further aspect of the invention is the use of the antibodies directedto the polypeptides of the invention for passive immunization. One coulduse the antibodies described in the present application.

In this connection, another embodiment of the present invention relatesto a composition for preventing or treating such diseases or infections.The composition of the present invention advantageously comprises anacceptable carrier and a SP1 and/or SP2 polypeptide(s) of the invention.Alternatively, the composition of the invention can comprise an antibodyand/or a polynucleotide and/or an expression vector of the invention.

In a preferred embodiment, the composition of the invention furthercomprises an adjuvant. As used herein, the term “adjuvant” means asubstance added to the composition of the invention to increase thecomposition's immunogenicity. The mechanism of how an adjuvant operatesis not entirely known. Some adjuvants are believed to enhance the immuneresponse (humoral and/or cellular response) by slowly releasing theantigen, while other adjuvants are strongly immunogenic in their ownright and are believed to function synergistically. Known adjuvantsinclude, hut are not limited to, oil and water emulsions (for example,complete Freund's adjuvant and incomplete freund's adjuvant),Corytzebactei-ium parvuin, Quil A, cytokines such as IL12,Emulsigen-Plus®, Bacillus Calmette Guerin, aluminum hydroxide, glucan,dextran sulfate, iron oxide, sodium alginate, Bacto Adjuvant, certainsynthetic polymers such as poly amino acids and co-polymers of aminoacids, saponin, paraffin oil, and muramyl dipeptide. Adjuvants alsoencompass genetic adjuvants such as immunomodulatory molecules encodedin a co-inoculated DNA, or as CpG oligonucleolides. The coin-oculatedDNA can be in the same plasmid construct as the plasmid immunogen or ina separate DNA vector.

Yet, a further embodiment of the present invention is to provide amethod for treating and/or preventing a Streptococcus suis-associateddisease or infection in an animal. The method of the invention comprisesthe step of administering to the animal a composition according to theinvention.

Further agents can be added to the composition of the invention. Forinstance, the composition of the invention may also comprise agents suchas drugs, immunostimulants (such as α-interferon, β-interferon,γ-interferon, granulocyte macrophage colony stimulator factor (GM-CSF),macrophage colony stimulator factor (M-CSF), and interleukin 2 (IL2)),antioxidants, surfactants, flavoring agents, volatile oils, bufferingagents, dispersants, propellants, and preservatives. For preparing suchcompositions, methods well known in the art may be used.

The amount of the components or the elements of the composition of theinvention is preferably a therapeutically effective amount. Atherapeutically effective amount of the contemplated component is theamount necessary to allow the same to perform their immunological rolewithout causing overly negative effects in the host to which thecomposition is administered. The exact amount of the components to beused and the composition to be administered will vary according tofactors such as the type of condition being treated, the type and age ofthe animal to be treated, the mode of administration, as well as theother ingredients in the composition.

The composition of the invention may be given to an animal throughvarious routes of administration. For instance, the composition may beadministered in the form of sterile injectable preparations, such assterile injectable aqueous or oleaginous suspensions. These suspensionsmay be formulated according to techniques known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparations may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents.They may be given parenterally, for example intravenously,intramuscularly or sub-cutaneously by injection, by infusion or per os.Suitable dosages will vary, depending upon factors such as the amount ofeach of the components in the composition, the desired effect (short orlong term), the route of administration, the age and the weight of theanimal to be treated. Any other methods well known in the art may beused for administering the composition of the invention.

4. Methods of Detection or Diagnosis and Kits

The SP1 and/or SP2 polypeptides, polynucleotides encoding same andantibodies of the invention may also be used in different ways in thedetection and diagnosis of Streptococcus suis-associated diseases orinfections caused by S. suis.

In this connection and in a further embodiment, the present inventionprovides a method for detecting the presence or absence of aStreptococcus suis strain in a sample, comprising the steps of:

-   -   a) contacting the sample with an antibody of the invention as        defined above for a time and under conditions sufficient to form        a complex; and    -   b) detecting the presence or absence of the complex formed in        a).

As used herein, the term “sample” refers to a variety of sample typesobtained from an animal and can be used in a diagnostic or detectionassay. The definition encompasses blood and other liquid samples ofbiological origin, solid tissue samples such as a biopsy specimen ortissue culture or cells derived therefrom.

Yet, in another embodiment, the present invention provides a method fordetecting the presence or absence of antibodies raised against aStreptococcus suis strain in a sample, comprising the steps of:

-   -   a) contacting the sample with a polypeptide of the invention as        defined above for a time and under conditions sufficient to form        an immune complex; and    -   b) detecting the presence or absence of the immune complex        formed in a).

One skilled in the art will recognize that this diagnostic test may takeseveral forms, including an immunological test such as an enzyme-linkedimmunosorbent assay (ELISA) or a radioimmunoassay, essentially todetermine whether antibodies specific for the protein (such as SP1and/or SP2) are present in an organism.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain an antibody or fragment thereof thatspecifically binds to a SP1 or SP2 polypeptide of the invention. One ormore additional containers may enclose elements, such as reagents orbuffers, to be used in the assay.

In this connection, the present invention also provides a diagnostic kitfor the detection of the presence or absence of antibodies indicative ofStreptococcus suis strain, comprising:

-   -   a SP1 and/or SP2 polypeptide according to the invention;    -   a reagent to detect polypeptide-antibody immune complex;    -   a biological reference sample lacking antibodies that        immunologically bind with said peptide; and    -   a comparison sample comprising antibodies which can specifically        bind to said peptide;        wherein said polypeptide, reagent, biological reference sample,        and comparison sample are present in an amount sufficient to        perform said detection.

Another diagnostic kit preferably contemplated is a kit for thedetection of the presence or absence of polypeptides indicative ofStreptococcus suis strain, comprising:

-   -   an antibody according to the invention;    -   a reagent to detect polypeptide-antibody immune complex;    -   a biological reference sample polypeptides that immunologically        bind with said antibody; and    -   a comparison sample comprising polypeptides which can        specifically bind to said peptide;        wherein said antibody, reagent, biological reference sample, and        comparison sample are present in an amount sufficient to perform        said detection.

EXAMPLES

The present invention will be more readily understood by referring tothe following examples. These examples are illustrative of the widerange of applicability of the present invention and are not intended tolimit its scope. Modifications and variations can be made thereinwithout departing from the spirit and scope of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice for testing of the present invention,preferred methods and materials are described hereinafter.

Example 1 Identification of a Surface Protein of Streptococcus suis andEvaluation of its Immunogenic and Protective Capacity in Pigs

A new Streptococcus suis surface protein reacting with a convalescentserum from pigs clinically infected by S. suis type 2 was identified.The apparent 110 kDa protein designated SP1 exhibits typical features ofmembrane-anchored surface proteins of Gram-positive bacteria such as asignal sequence and a LPVTG (SEQ ID NO: 10) membrane anchor motif.Moreover, a conserved avirulence domain that often found in plantpathogens has been detected. Electron microscopy using a SP1-specificantiserum has confirmed the surface location of SP1 protein on S. suis.The SP1-specific antibody reacts with the cell lysates of most S. suisserotypes and type 2 isolates in immunoblots, demonstrating its highconservation in S. suis species. Immunization of piglets with therecombinant SP1 by intramuscular route elicits a significant totalimmunoglobulin G (IgG) antibody response. However, the antibody responseis not reflected in protection of pigs that are intratracheallychallenged with a virulent strain in our conventional vaccination model.

Materials and Methods

Bacterial strains, phage, plasmids and media. Reference strain S735 ofS. suis serotype 2 was used for the genomic library construction.Reference strains of the thirty-three serotypes (types 1 to 31, 33 and1/2), 26 field strains of serotype 2 from different origin as well asfive other Gram-positive organisms are listed in Table 1. Phage LambdaZap II and Escherichia coli XL1-Blue MRF strain were obtained from acommercial source (Stratagene, La Jolla, Calif.). S. suis were grown inTodd-Hewitt broth (THB, Difco, Detroit, Mich.) or agar plates (QuelabLaboratories, Montreal, Canada) at 37° C. with 5% of CO₂, while otherGram-positive bacteria were grown as recommended by the ATCC catalogue.E. coli was grown in either Luria-Bertani (LB) medium alone or LB mediumsupplemented with 2 g of maltose/liter at 37° C. Where appropriate, E.coli was grown in the presence of 50 μg of ampicillin/ml and 0.8 mMisopropyl-β-D-thiogalactopyranoside (IPTG). pMal™-p vector (New EnglandBioLabs) was used for generating the MBP-SP1 fusion protein.

Antisera. Convalescent swine sera were collected from pigs clinicallyinfected with S. suis type 2 strain S735. Monospecific anti-SP1 serumwas obtained by immunizing New Zealand White rabbits intravenously with230 μg of purified SP1 emulsified with 0.5 ml of Freud's incompleteadjuvant. The rabbits received two booster injections with the same doseof the SP1 at 2-week intervals and then were bled 10 days after the lastbooster immunization. Sera were stored at −20° C. until used.

Identification, cloning, and sequencing of the sp1 gene. Chromosomal DNAfrom S. suis S735 strain was isolated as previously described (33).Purified chromosomal DNA was partially digested with the restrictionenzyme EcoRI, and the resulting fragments were electrophoresed in 1%agarose gel. Fragments in the 6- to 10-kb size range were extracted fromthe gel and ligated to the EcoRI arms of λZAPII vector, and the vectorwas encapsidated using the Gigapack II packaging extract (Stratagene).The recombinant phages were used to infect E. coli XL1-Blue MRF′. whichwas then plated onto LB agar. The resulting plaques were lifted ontonitrocellulose membranes (Bio-Rad, Mississauga, Ontario, Canada). Themembranes were blocked using Tris-saline buffer (TBS) with 2% skim milkand sequentially incubated with the convalescent swine serum from S.suis serotype 2 infection, peroxidase-conjugated rabbit anti-swineimmunoglobulin G (IgG) antisera (Jackson Immuno Research Laboratories,Inc., West Grove, Pa.), and O-phenylenediamine. The positive plaqueswere purified to homogeneity. The recombinant pBluescript plasmids wereexcised with ExAssist helper phage (Stratagene) according to themanufacturer's instructions. The sequence of the insert was determinedusing T3 and T7 promoters as primers in DNA Sequencing Facility,University of Maine (Orono, Me., USA). The nucleotide and amino acidsequences deduced from open reading frames (ORFs) were analyzed usingprograms available on the internet.

The sequence coding for mature SP1 was amplified from purifiedchromosomal DNA of strain S735 by PCR primers P1(5′-ATGGATCCATTGAAGGCCGCTCGGCACAAGAAGTAAAA-3′; SEQ ID NO 13) and P2(5′-CCAAGTCGACTTATAATTTACGTTTACGTGTA-3′; SEQ ID NO 14), which containedBamHI and Sal I restriction sites, respectively. The PCR was performedwith 5 min at 94° C., followed by 30 cycles of 1 min at 94° C., 30 s at56° C., and 1 min at 72° C. The resulting PCR fragment was cloned intoBam HI and Sal I sites of pMAL-p expression vector. The recombinantplasmid containing the sp1 gene was named pORF3.

Expression and purification of recombinant SP1 protein. The purifiedplasmid pORF3 was used to transform E. coli XL1-Blue strain byelectroporation with Genepulse II apparatus (Bio-Rad) following themanufacturer's recommendations. This recombinant strain was grown in LBmedium plus 2 g of glucose/L and 50 μg of ampicillin/ml. Forover-expression, the culture was inoculated from an overnight culturewith its starting OD₆₀₀ adjusted to 0.1. The culture was incubated withagitation until OD₆₀₀ of approximately 0.8, and then IPTG was added inorder to induce production of the MBP-SP1 fusion protein. After 2 hoursof the induction, the fusion protein was found in the bacterialperiplasm as well as in the cytoplasm. It was decided to use extracts ofthe bacterial lysates for purification of the SP1 protein.

The fusion protein was purified by affinity chromatography using anamylose resin (New England BioLabs) following the manufacturer'sinstructions. The E. coli cell pellet was suspended in the affinitycolumn binding buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4) and cellswere lysed using the French Pressure Cell Press (SLM Instruments, Inc.).After filtration with a 0.45 μm membrane, the supernatant was subjectedto the amylose resin. The MBP-SP1 fusion protein was eluted with 1%maltose in the binding buffer and protein-containing fractions weredetermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The purified fusion protein was cleaved with protease FactorXa (New England BioLabs) at a concentration of 20 μg/mg protein, andapplied to a mono-Q column (Amersham Pharmacia Biotech, Baie d'Urfee,Canada). The recombinant SP1 devoid of MBP carrier was eluted from thecolumn by using a linear NaCl gradient (0 to 0.4 M NaCl in 20 mMTris-HCl, pH 7.4). The SP1-containing fractions were combined anddialyzed against PBS buffer. The purity of the recombinant SP1 wasevaluated by SDS-PAGE, and the concentration of the protein wasdetermined by the Bradford protein assay (Bio-Rad) according to themanufacturer's instructions.

SDS-PAGE and western immunoblotting. SDS-PAGE was performed as describedby Laemmli (21). Total cell extract or purified protein was separated ona 10% acrylamide gel and the gel was then stained with Coomassiebrilliant blue R250 (Sigma, St. Louis, Mo.). Prestained low molecularmass markers (Bio-Rad) were used to determine the apparent molecularweights of proteins. Alternatively, Western blotting of proteinstransferred to nitrocellulose membranes was performed essentially asdescribed by Burnette (5).

Immunoelectron microscopy. S. suis S735 strain was grown in 5 ml of THBovernight, centrifuged, and resuspended in 500 μl of PBS (pH8.0). 20 μlof the bacterial suspension was placed on nickel-formvar grids (INRS,Institut Armand Frappier, Laval, Canada) and allowed to partially airdry. After blocking for 30 min with 10% normal donkey serum in dilutionbuffer (PBS-1% bovine albumin-1% Tween 20, pH8.0), the grids were soakedin 50 μl of SP1-specific rabbit serum or control rabbit anti-MBP serum(New England BioLabs) diluted 1/25 in the dilution buffer for 2 h atroom temperature. The grids were washed three times in PBS-1% Tween20,and then transferred into 50 μl of 12 nm colloidal gold-affinipuredonkey anti-rabbit IgG (Jackson Immuno Research Laboratories) diluted1/30 in the dilution buffer and incubated for 1 h at room temperature.After three washes with PBS-1% tween20 and one wash with distilledwater, bacteria were stained with 1% phosphotungstic acid and examinedwith an electron microscope (Philips 201) at an accelerating voltage of60 kV.

Immunization and protection study. Pigs were used to perform theimmunization and protection assay at VIDO (Saskatoon, Canada) inaccordance with principles outlined in the “guide to the care and use ofexperimental animals” of the Canadian Council on Animal Care using aprotocol that was approved by the University Committee on Animal Care(37). Three week-old piglets with average weight of 8.23 kg from a herdthat is free of S. suis serotype 2 were randomly assigned to two groupsof eight. The pigs were injected intramuscularly twice at a 3-weekinterval with 1 ml of either 100 μg purified SP1 mixed with 30%Emulsigen-Plus (MVP Laboratories, Ralston, Nebr.) adjuvant or 30%Emulsigen-Plus in physiological saline as a control. Eleven days afterthe second injection, the immunized and control animals were challengedby aerosol of 1 ml (4.6×10⁶ CPU) of a log-phase culture of S. suisvirulent strain 166, which has been confirmed to be highly virulent (3).Blood samples were collected prior to each injection, challenge and theend of the experiment for determination of antibody responses. Pigs weremonitored daily for clinical signs, body temperature and mortality forten days after challenge. All pigs were examined postmortem for grosspathology and blood was cultured to detect the presence of S. suisbacteremia.

ELISA. Serum SP1-specific total IgG and IgG isotypes (IgG1 and IgG2) ofimmunized piglets were determined by enzyme-linked immunosorbent assay(ELISA). Polysorb plates (Nunc-Immunoplates, Rochester, N.Y., USA) werecoated overnight at 4° C. with 100 μl per well of the purifiedrecombinant SP1 at a concentration of 0.3 μg/ml in carbonate buffer.After three washes with PBS containing 0.05% Tween20 (PBST), the plateswere blocked with 5% skim milk in PBST for 1 h at 37° C. fordetermination of total IgG, swine sera from the control and vaccinegroups were diluted 1/5000 in PBST and added to appropriate wells induplicate at 100 μl per well. After incubation for 1 h at 37° C. andwashing three times, bound antibodies were detected by incubation for 1h at 37° C. with peroxidase-conjugated goat anti-swine IgG(H+L) antisera(Jackson Immuno Research Laboratories). For IgG1 and IgG2 detection,1/500 diluted swine sera from vaccine group were added at 100 μl perwell. Mouse anti-porcine IgG1 or IgG2 (Serotec, Kidlington, Oxford, UK)was used as the primary antibody, and peroxidase-conjugated goatanti-mouse IgG(H+L) (Serotec) was used as the secondary antibody. Theplates were developed with TMB substrate (Zymed, S. San Francisco, USA).Absorbance was measured at 450 nm in an ELISA reader (Power Wave 340,Bio-Tek Instruments, Inc.). Results were expressed as the means±S.D.Statistical significance was determined by Student's t test.

Nucleotide sequence accession number. The sequence of the gene encodingSP1 protein of S. suis is shown in FIG. 2 and has been assigned GenBankaccession number AY864331.

Results

Identification of sp1 gene. The S. suis chromosomal library wasconstructed from the S. suis S735 strain in λZAPII and screened usingconvalescent swine sera from S. suis serotype 2 infected animals. Oneclone, which expressed a protein with an apparent molecular weight (MW)of 110 kDa that was strongly reactive against the convalescent swineserum, was selected for further characterization. The recombinantpBluescript plasmid, designated pSS735, was excised from thebacteriophage arms, and its schematic organization is presented inFIG. 1. DNA sequence analysis of the 6057-bp insert of the pSS735revealed four ORFs. This gene cluster was found in the partiallysequenced genomes of S. suis Canadian strain 89/1591 (NZ_AAFA00000000)and European strain P1/7 (NC_004549) with the same organization. Thededuced amino acid sequences of both ORF1 and ORF2 showed identitiesranging from 50-80% with a glycosyl transferase, and ORF4 showedidentities ranging from 50-75% with a catabolite control protein A frommany bacterial species, most of them belong to the genus Streptococcus.The ORF3 encodes a 670 amino acid protein, designed SP1, with apredicted pl of 6.0 and a calculated molecular mass of 74.8 kDa.Comparison of the amino acid sequence of SP1 with those in availabledatabases revealed no significant homology with other proteins.Subcloning analysis of the sp1 sequence in pMal-p vector revealed thatthe SP1 strongly reacted with the convalescent swine serum,demonstrating that SP1 is the immunogenic protein.

SP1 is a novel C-terminal-anchored surface protein of S. suis. The 2010bp of sp1 gene starts with an ATG codon which is preceded by putativeShine-Dalgarno sequence (GAAAGGA) 10 bp upstream of the start codon andterminates with a TAA codon (FIG. 2). Analysis of the predicted SP1amino acid sequence revealed a hydrophobic core of 15 amino acids at theN-terminus and a putative signal-peptidase cleavage site between Ala²⁹and Gln³⁰. Ten repeats of 27-amino-acid sequence with a strong consensuspattern, separated by 3-amino-acid residue spacers, were detected withinthe carboxyl half of the protein. Immediately C-terminal from the repeatregion is a cell wall-associated region, which spans 49-amino-acidresidues and is characterized by a high percentage of threonine residues(20.4%). This threonine-rich region is immediately followed by an LPVTG(SEQ ID NO: 10) consensus motif typical of membrane-anchored surfaceproteins of many Gram-positive bacteria. Beginning four amino acidsC-terminal from the membrane anchor motif, a second hydrophobic segmentof 16 amino acids was identified, which is followed by four positivecharged amino acid residues at the C-terminal end of the protein (FIG.2).

Analysis of the amino acid composition revealed a region of absence ofaromatic residues between Glu²⁷² and Thr⁶³⁰, which spans all of therepeat sequences. Furthermore, a conserved domain search using BLASTidentified an avirulence domain in Lys³¹⁹ to Val⁶⁰¹ region, whichexhibits similarity with AvrXa7 avirulence factor from the plantpathogen Xanthomonas oryzae pv. oryzae (41) with 20% identity (FIG. 3).If conservative amino acid substitutions are taken into consideration,the similarity is 48%.

Production of the recombinant SP1. The sequence coding for mature SP1protein was amplified by PCR and ligated into the IPTG-inducible pMAL-pvector. The resulting recombinant plasmid was expressed in E. coliXL1-Blue strain. As shown in FIG. 4A, induction of the E. colirecombinants harboring the malE-sp1 fusion gene led to the expression ofan approximately 150 kDa of MBP-SP1 fusion protein (lane 2) which wasabsent in the non-induced E. coli cells (lane 1). The fusion protein wasmostly found in the cytoplasm of the E. coli cells (lane 3).Interestingly, a truncated MBP-SP1 fusion protein in which the repeatingregion characterized by absence of aromatic substitutions was deletedwas completely transported into the periplasmic space (data not shown),suggesting that this region somehow interfered with MBP localization.

The fusion protein was purified by using affinity matrix amylose columnand eluting with maltose, and showed a single protein band ofapproximate 150 kDa on SDS-PAGE (lane 4). The purified fusion proteinwas proteolytically cleaved with Factor Xa, yielding the apparent 110kDa of mature SP1 and the expected 45 kDa of MBP tag (lane 5). Themature SP1 devoid of MBP was obtained by subsequent purification withion-exchange chromatography, with a purity>95% estimated by SDS-PAGE(lane 6). Both the MBP-SP1 fusion protein and the purified recombinantSP1 demonstrated specific reactivity in a western blot to theconvalescent swine serum used for the initial screening of the genomiclibrary (FIG. 4B). Identity of the purified SP1 was confirmed byN-terminal protein sequencing. The protein concentration was measuredwith Bradford protein assay and adjusted to 1 mg/ml.

Cell surface expression of SP1 in S. suis. To confirm the location ofSP1 on the surface of S. suis cells, immunoelectron microscopy wasconducted by using a monospecific polyclonal anti-SP1 antibody, R44.Immunogold particles were found to be evenly distributed on the surfaceof the S. suis S735 strain. This indicates that SP1 protein ishomogeneously expressed on the cellular surface. Rabbit anti-MBP serumwas used as control and did not show any labeling (FIG. 5).

Distribution of the SP1 among S. suis. To evaluate the conservation ofSP1 among reference strains of deferent serotypes of S. suis andserotype 2 field strains, whole cell preparations of the bacteria wereapplied to western blots and detected by SP-specific antibody R44. Asshown in Table 1, except for strains of serotypes 13, 16, 20, 22 and 24,R44 reacted with other 28 S. suis serotypes, while 25 of 26 tested type2 isolates from different geographic origins reacted with the R44antibody. Five strains from other species of Streptococci were used toverify the specificity of SP1 and no SP1 protein were detected.

Immunogenicity of SP1 and protection of pigs against challenge with S.suis. Groups of 8 piglets were immunized twice intramuscularly witheither 100 μg of purified recombinant SP1 emulsified with the adjuvantor adjuvant only. Immunization of pigs with SP1 triggered anantigen-specific response (FIG. 6A). Analysis of corresponding seraobtained from the control animals and the animals before immunizationclearly indicated that there was no SP1-specific antibody, since onlybackground ELISA values were recorded. Only two weeks after the firstinjection, SP1 elicited a significant IgG response that was obviouslyenhanced by the boost immunization. Assessment of IgG isotypesdemonstrated that sera from immunized pigs contained both IgG1 and IgG2antibodies (FIG. 6B). However, IgG1 response dominated over IgG2,suggesting that vaccination with SP1 mainly induced the Th2-like immuneresponse. Aerosol challenge of the pigs with S. suis 166 strain resultedin steady increases of clinical score starting from day 2 after thechallenge and there was no significant effect of the vaccination. Assummarized in Table 2, although fewer pigs suffered arthritis in thevaccinated group than in the control group, both groups showed similarsymptoms after challenge. Three pigs from each group died or wereeuthanized due to high clinical scores prior to the end of theexperiment. S. suis bacteremia was found in all dead pigs and was notdetected in the surviving pigs.

Discussion

First immunization of pigs elicited rapid SP1-specific humoral antibodyresponse that can be significantly boosted by a subsequent injection.However, the antibody to SP1 did not confer immunity against anheterologous challenge using S. suis strain 166. This discrepancybetween antibody response and protection have been reported in someother surface antigens of Gram-positive bacteria, such as streptococcalfibronectin binding protein (Sfb1) (26), pneumococcal surface protein A(PspA) (27), group B polysaccharide (25) and M-like protein (SeM) ofStreptococcus equi (34). The reason why antibodies against SP1 were notprotective against the challenge by S. suis 166 is unclear. In aphagocytic killing study, presence of pooled serum from theSP1-immunized pigs did not promote S. suis killing by porcineneutrophils, suggesting that the antibodies are lacking opsonophagocyticfunction. Host protection against infection caused by S. suis, a highlyencapsulated microorganism, is mediated primarily by phagocytosis (32).Therefore, total IgG levels generated in the Applicant's conventionalvaccination model may not adequately reflect the presence of protectiveantibodies that are capable of triggering leukocyte effector functions.To further illustrate the immune response types trigged by SP1, IgGisotypes in immunized sera were assessed. IgG1 levels were consistentlyhigher then IgG2 levels suggesting the induction of Th2-like responses.Although the concept of “Th1/Th2” balance is not yet well documented inpigs as in some other species, recent evidence showed that porcine IgG2had greater complement activating ability than did IgG1 (6).

Emulsigen-Plus was used as an adjuvant in this study, because it wasbelieved to be capable of creating an antigen depot at the site ofinoculation from which the antigen is slowly released and thus providingprolonged stimulation to the immune system (23, 37). However, recentevidence showed that vaccine formulated with Emulsigen alone triggeredpredominantly an IgG1 response but very weak Th1-type immune response(19, 28). Evidence from vaccination using surface antigens of otherGram-positive bacteria has demonstrated that efficiency ofopsonophagocytosis can be dramatically enhanced by using Th1-directingadjuvants, such as CpG and interleukin-12 (IL-12) (4, 22, 24). Theseadjuvants promote a Th1-type immune response characterized by enhancedproduction of opsonizing antibodies, specially IgG2 isotype.Furthermore, the enhanced antibody-mediated opsonization was clearlyreflected in protection (2, 40).

In conclusion, SP1 is a novel C-terminal-anchored surface protein of S.suis, as demonstrated by analysis of the molecular features and electronmicroscopy. Vaccination with the recombinant SP1 elicited significanthumoral antibody response in piglets, along with the fact thatconvalescent swine sera present high titers of antibody against thisprotein, suggesting that SP1 is an exposed antigen of S. suis. Takentogether with its wide distribution in different S. suis serotypes,these findings made the SP1 a candidate for consideration in thedevelopment of a subunit vaccine. The potential of SP1 as a vaccinecandidate will be demonstrated in the following Examples.

Example 2 Recombinant SP1 Protects Mice Against S. suis ChallengeInfection

This study is to evaluate whether the SP1 recombinant protein isprotective as a subunit vaccine candidate in a mouse model with amodified immunization route and adjuvant.

EXPERIMENTAL PROCEDURE: Mice (CD1) were randomly assigned to two groupsof ten, and immunized subcutaneously twice at 2-week interval witheither 20 μg of purified SP1 mixed with 20 μg of Quil A as a adjuvant or20 μg of Quil A only as a control (Table 1). Ten days after the secondvaccination, the animals were challenged i.p. with 1×10⁸ CFU of a S.suis virulent strain (31533). The mice were monitored twice a day forclinical signs and mortality until day 14 after the infection. Bloodsamples were collected prior to each vaccination and challenge fordetermining antibody responses.

RESULTS: Vaccination with SP1 elicited significant humoral IgG responsesin mice after primary immunization (mean titre 3×10⁴) and a boosterinjection significantly increased the antibody titre (1.8×10⁶). Incontrast, the SP1-specific IgG in sera of control group was atundetectable level (FIG. 1). Furthermore, all of four IgG subclasseswere induced in SP1-immunized mice, with the IgG2a titre being thehighest (1.75×10⁶) followed by IgG1 (1.2×10⁶), IgG2b (7.25×10⁵) and IgG3(3.7×10⁴) (FIG. 2). Specificity of SP1-induced antibodies wasdemonstrated by Western blot in which pooled sera collected fromSP1-immunized mice can recognize the purified SP1 and the SP1 protein inS. suis S735 and 31533 cell preparations.

Sixteen hours after administering the challenge infection, all mice incontrol group started to exhibit clinical signs (septicemia), such asthe ruffled hair coat (suggesting fever) and slow response to stimuli.Starting from day 4 after the challenge, 8 of 10 mice in this groupsuccessively developed severe central nervous system symptoms(meningitis) such as running in circles and opisthotonos. All of the 8ill mice died, or had to be euthanized due to the severity of thecondition. In contrast, except for 6 of 10 mice in SP1-vaccinated grouphad transient clinical signs such as slight rough hair and reluctant tomove during 16-40 hours after the challenge, all mice in this groupremained healthy during the observation period (FIGS. 3 and 4).

DISCUSSION AND CONCLUSION: The difference in protection observed inmouse and pig models is explained by well-balanced IgG subclass levelsevoked in the mouse vaccination model, especially the extremely highIgG2a titre. Among murine antibody isotypes, IgG2a has been shown to bethe most effective at activating opsonophagocytic function of leukocytes(2, 42, 43)). Furthermore, S. suis, an encapsulated bacterial, is mosteffectively eliminated by opsonophagocytosis. Thus, it is likely thatpredominant IgG2 production contributed most to the observed protection.Nevertheless, these data indicate that immunization of mice with SP1 byusing a Th1 inducing adjuvant, such as Quil A, can induce an efficientantigen-specific response, and protect mice against challenge infectionwith a lethal dose of a virulent S. suis strain and result in completeprotection from S. suis death (FIG. 4).

Example 3 Identification of a Novel Gene Encoding a Streptococcus suisProtein with IgG-Binding Activity and Protective Capacity

In the Applicant's continued effort to understand the pathogenicmechanism of S. suis and search for its protein(s) that may be useful inthe development of a reliable diagnostic reagent or vaccine, a newprotein which exhibits IgG-binding activity was identified from avirulent strain of S. suis serotype 2. This apparent 58-kDa proteindesigned SP2 contains a 23 amino acids cleavable N-terminal signalsequence and a lysine M motif near the N-terminus, and six identicalrepeats of 13 amino acids each within the C-terminal part. SP2 is highlyconserved among different S. suis serotypes as demonstrated by PCRamplification of the SP2 gene. Recombinant SP2 strongly reacted with aconvalescent swine serum collected from pigs clinically infected by S.suis type 2. Immunization of mice with the purified recombinant SP2elicits a significant antibody response that conferred a partialprotection against challenge infection with a virulent S. suis strain.

These results show that SP2 is a potential diagnostic agent and vaccinecandidate for S. suis infection.

Experimental Procedures and Results

Identification of SP2 Gene

A positive phage which reacted by non immune mechanism with differentclasses and species of Ig (Pig IgG, Human IgG and IgA) was identified byscreening the constructed S. suis serotype 2 genomic library. Sequenceof the DNA insert revealed a 6.3 kb insert which contains three ORFscoding for dehydrogenases, SP2 and dextran glycosidases (44),respectively (FIG. 12). This gene cluster was found in the partiallysequenced genomes of S. suis Canadian strain 89/1591 (NZ_AAFA00000000)and European strain P1/7 (NC_004549) with the same organization. The SP2amino acid sequence presented similarities with some streptococcalproteins usually exhibiting Ig-binding activity. An identity of 45% in a395-amino acid stretch was observed with a Conserved hypotheticalprotein of Streptococcus pneumoniae (AAL00677). Other homologies werefound with a putative 42 kDa protein of Streptococcus pyogenes (45%identity over 388-amino acid stretch) (AAK33481) and with a group Bstreptococcal surface immunogenic protein (40% identity over 434-aminoacid stretch) (60) (AAG 18474).

Characterization of SP2 Protein

The 1158 bp SP2 gene encodes a 386-aa SP2 protein, with a theoretical plof 4.40 and molecular mass of 42.5 kDa. This protein was rich in valine(15%), glutamic acid (10%), and alanine (9%). Charge distributionanalysis of SP2 revealed one positive charge cluster (K²-K²⁶) at theN-terminus and one negative charge cluster (D¹⁶⁸-E²⁴²) in middle of theprotein (FIG. 13). The positive charge cluster was followed by aputative signal sequence of 23 amino acids. The amino acid sequence ofSP2 contains a LysM (lysine) motif at positions 71 through 109. ThisLysM domain is found in a variety of enzymes involved in bacterial cellwall degradation and has a general peptidoglycan binding function,suggesting that SP2 may be a surface protein of S. suis. Thus, theN-terminal constitution of SP2 outlined a possibility that the positivecharge cluster remained in the cytoplasm functions as a temporary stopand helps in formation of mature SP2 by cleaving the signal sequence andin location of SP2 on the bacterial surface via binding of LysM domainto peptidoglycan. Furthermore, six identical repeating sequences of 13amino acids were identified in the middle part of SP2 (FIG. 13).

Distribution of SP2 Gene in Different S. suis Serotypes

To evaluate the conservation level of SP2, PCR were performed usingprimers covering the full-length SP2 gene. PCR was performed with aninitial denaturing at 94° C. for 5 min followed by 30-cycles of 1 min at94° C., 1 min at 52° C. and 2 min at 72° C., and a final elongationperiod of 10 min at 72° C.

The forward and reverse primers used for SP2 distribution in differentserotypes were respectively:

(5′-TTTAAAAGAACGGTTGAAGGC-3′; SEQ ID NO: 15) and5′-GCATAAGCTGCCACTTGATCT-3′; SEQ ID NO: 16).

SP2 gene was amplified from 31 of the 33 serotype reference strains withsome size variations (FIG. 14). Sequence analysis of selected variantfragments suggested that the number of repeats in the SP2 gene isresponsible for the size variations.

Production and Purification of Recombinant SP2

The gene coding for mature SP2 was generated by PCR from S. suis S735chromosome and subcloned to a pET32+ vector (New England BioLabs). Theconstruct was used to transform E. coli DE3 strain by electroporationwith Genepulse II apparatus (Bio-Rad) following the manufacturer'srecommendations. For over-expression, the culture was inoculated from anovernight culture with its starting OD₆₀₀ adjusted to 0.1. The culturewas incubated with agitation until OD₆₀₀ of approximately 0.8, and thenIPTG (0.5 mM) was added in order to induce production of the Trx-His-SP2fusion protein. After 2 hours of the induction, bacterial cytoplasm wereprepared and used for purification of the SP2 protein.

The Trx-His-SP2 fusion protein was purified from the cytoplasm byaffinity chromatography using Ni+ column (Amersham Pharmacia Biotech,Baie d'Urfee, Canada). The cytoplasm was filtered with a 0.45 μmmembrane and subjected to the column. The fusion protein was eluted with500 mM imidazole in binding buffer and protein-containing fractions weredetermined by SDS-PAGE. The purified fusion protein was cleaved by0.001% (w/w) of enterokinase (New England BioLabs), yielding an apparent58 kDa SP2 and the expected 20 kDa Trx-His tag (FIG. 15), and thenapplied to a mono-Q column (Amersham Pharmacia Biotech, Baie d'Urfee,Canada). The recombinant SP2 devoid of Trx-His tag was obtained fromedition of the column by using a linear NaCl gradient, with an estimatedpurity greater than 95% as visualized by SDS-PAGE (FIG. 15). The proteinconcentration was determined by the Bio-Rad protein assay kit (BioRad)according to the manufacturer's instructions. Identity of the purifiedSP2 was confirmed by N-terminal protein sequencing.

SP2 is an Immunogenic Protein of S. suis and Exhibits IgG-BindingActivity

SP2-specific antibody was generated by immunizing New Zealand Whiterabbits intramuscularly with 100 μg of recombinant SP2 proteinemulsified with 0.5 ml of Freud's incomplete adjuvant. The rabbitsreceived two booster injections with the same dose of the SP2 at 2-weekintervals and then were bled 10 days after the last boosterimmunization. The SP2 specific antibody conversely recognized SP2 in S.suis cell preparation in a western blot (FIG. 16a). Moreover,recombinant SP2 reacted with a convalescent swine serum (FIG. 16b),demonstrating that the anti-SP2 antibody exists in the serum of pigsclinically infected by S. suis.

The binding activities of the recombinant SP2 to human and pig IgG weredemonstrated in FIGS. 16c and 16d.

Recombinant SP2 Partially Protects Mice Against S. Suis ChallengeInfection

Mice (CD1) were randomly assigned to two groups of eleven (vaccinegroup) and ten (control), and immunized subcutaneously twice at 2-weekinterval with either 50 μg of purified SP2 mixed with 20 μg of Quil A asa Th1 inducing adjuvant or 20 μg of Quil A only as a control. Ten daysafter the second vaccination, the animals were challenged i.p. with1×10⁸ CFU of a S. suis virulent strain (31533). The mice were monitoredtwice a day for clinical signs and mortality until day 14 after theinfection. Blood samples were collected prior to each vaccination andchallenge for determining antibody responses.

Vaccination with SP2 elicited significant humoral IgG responses in mice.In contrast, the SP2-specific IgG in sera of control group was atundetectable level (FIG. 17). Both groups showed clinical signs ofsepticemia and meningitis, however, the clinical scores in SP2vaccination group are lighter than in the control group (FIG. 18). 4 of11 mice in SP2 vaccination group died or had to be euthanized due to theseverity of the condition (survivor rate=64%). In contrast, 8 of 10 micein control group died (survivor rate=20%) (FIG. 19). These results showthat SP2 protects mice against S. suis challenge infection.

Conclusion

SP2 is a new described S. suis immunogenic protein which shares littleidentity with oilier known sequences. Convalescent swine sera presentantibody against this protein, demonstrating that SP2 is a potentantigen that is expressed during S. suis infection. These findings,along with its wide distribution in different S. suis serotypes, makethe SP2 a candidate for consideration in the development of a diagnosticreagent. Since vaccination of mice with recombinant SP2 resulted inprotection, it is thus clear that SP2 is a potential vaccine candidateagainst S. suis infection.

Example 4 Effect of Immunization of Piglets with ExperimentalStreptococcus Suis Vaccine

This study evaluates the protective effect of recombinant Sao protein onS. suis serotype 2 challenge infection in piglets.

Materials and Methods

Animals, Allocation to Treatment and Exclusion Criteria:

A total of 24 crossbred piglets from S. suis disease-free herd (H & MFast Farms Inc.) without any previous vaccination against S. suis wereused. The pigs were kept under commercial conditions at the herd oforigin from birth until they were weaned at an average weight of 7.79 kgat 23.5 days of age. Pigs were housed with controlled temperature (27 to30° C.) and ventilation, on vinyl-covered metal flooring, and wereprovided with water via nipple waterers and had free access tocommercially-prepared, nutritionally balanced, antibiotic-free feed. Aveterinarian examined the pigs prior to the beginning of the study. Allwere healthy. At weaning, the piglets were randomly assigned to twogroups, balanced by body weight.

Group 1: 200 μg Sao and 400 μg Quil A in 1 mL Saline

Group 2: 400 μg Quil A in 1 mL Saline (control)

Any animals that receive an unintended treatment or succumb to anunrelated disease will be excluded from analysis.

Vaccination and Challenge:

All pigs were vaccinated IM with 1 ml twice at 2-week interval. Bloodsamples were collected before each injection and challenge. There wereno adverse events as a result of these treatments.

Two weeks after the second vaccination, the pigs were anesthetized withhalothane and challenged by aerosol of 1 ml of a suspension of S. suis166. The bacteria were from a log-phase culture grown in filtersterilized Todd-Hewitt Yeast Broth to an OD₆₂₀ of 0.8 and diluted 1:100in saline (0.85% NaCl). The bacterial concentration administered to pigswas later measured to be 6.8×10⁶ CFU/ml.

Clinical Observations:

A veterinarian or trained animal care technician clinically evaluatedthe pigs once daily and measured body temperatures during assignment ofclinical scores each morning for ten days after challenge. A dailyclinical score (from 0 to 4) was derived as the sum of attitude andlocomotion scores for each animal based upon signs of nervous,musculoskeletal or respiratory disease as follows:

Attitude:

-   0=Normal attitude and response to stimuli-   1=Inactive and slow to respond; oculo-nasal secretions-   2=Only responsive to repeated stimuli, apathetic-   3=Recumbent, nonresponsive, unaware of surroundings-   4=Dead    Locomotion:-   0=Normal gait and posture-   1=Slight in coordination, lameness and/or joint swelling but rises    without assistance-   2=Clearly uncoordinated or lame but stands without assistance-   3=Severe lameness, severe ataxia, does not remain standing-   4=Dead

Pigs having a clinical score greater than 2 on either scale wereeuthanized by lethal injection. Pigs with rectal temperatures equal toor greater than 40.6° C. and a clinical score greater than 0, as well asthose pigs that were dead, were recorded as sick on that day. Pigs thatdied or were euthanized prior to the end of the experiment on day 9 wererecorded as dead for evaluation of the effect of treatment on mortalityrate. All individuals making judgements about animals, evaluatingclinical signs of disease, or performing laboratory assays were blind tothe identity of the treatment.

Haemotologic Condition:

A heparin-treated blood sample was obtained by venipuncture fordetection of S. suis bacteremia (by culture on days 0 and 3 afterchallenge and postmortem).

Antibody Titre:

Titers of Sao-specific total IgG and IgG subclasses (IgG1 and IgG2) insera were determined by ELISA. The serum dilution that resulted in anOD450 reading of 0.1 after background subtration was considered thetiter of this serum.

Necropsy:

All pigs were examined postmortem and the following tissues werecultured for bacteria: cerebellum swab, tracheobronchial lymph node, ajoint swab (an affected joint if lesions are present; otherwise a stiflejoint), and blood. The number of S. suis bacteria that were recoveredwas recorded on an ordinal scale from 0 to 4 (approximating the log₁₀number of colonies). In addition, the extent (percentage) of pulmonaryinvolvement was estimated by visual examination.

Statistical Analysis

The significance of differences between groups in nominal data(mortality, presence or absence of S. suis in the tissues, days sick orwell) was determined using contingency table analysis andLikelihood-Ratio Fisher Exact Test. The significance of differencesbetween groups in ordinal data (clinical score) was transformed byranking and determined by t-test. The significance of differencesbetween groups in survival curves was determined by survival analysisusing the logrank test (equivalent to the Mantel-Haenszel test). Thesignificance of differences among groups in continuous data (length ofsurvival after challenge, body temperature, log₂ CFU/ml of blood) wasdetermined using t-test (after appropriate transformation to normalityas required).

Results

Excluded Animals:

One pig was humanely killed on day 5 after the challenge because ofpersistent; worsening prolapsed rectum. This pig will be excluded fromanalysis.

Clinical Observations:

-   1. Response to immunisation: There were no unusual reactions    attributable to vaccination.-   2. Body temperature: The body temperature data as analysed by    t-test, showed no significant difference between the two group    (p>0.05). Generally the vaccinated pigs tended to have lower    temperatures (FIG. 20)-   3. Clinical disease: The “clinical score” is a measure of the amount    of disease and incorporates both mortality and morbidity. The two    groups were compared using Mann-Whitney analysis of an effect of    vaccine on clinical score, and the clinical disease in vaccinated    group was significantly less than that in control (p=0.024) (FIG.    21).-   4. Survival of pigs after S. suis challenge: The survival rate is    82% in vaccination group and 42% in control group, respectively.    Comparison of survival curves using the two data sets, shows that    the survival time of vaccinated pigs was significantly longer than    that of control (p=0.048) (FIG. 22).    Bacteriology-   1. Bacteremia: Bacteria in the blood of piglets were not detected    (ND) before challenge. The bacteremia pigs after challenge and    postmortem were not significant different between the two groups.    However, the vaccinated pigs had less bacteremia (table 3).-   2. Infection postmortem: Microbiologic culture of samples from the    brain, tracheobronchial lymph node, and joint of all of the pigs    that were challenged was done to monitor the level of infection.    Number of tissues from that bacteria with colonial morphology    typical of the challenge strain were recovered was shown in table 4.    The Wilcoxon Rank Sum test for the effect of vaccinated group on the    median bacteriology score (median of the sum of scores for all    tissues of each pig) showed that this difference was significant    (p=0.007).    Pathology (Post-Mortem)

Pathologic lesions of arthritis or pneumonia were detected in only 6pigs (2 in vaccinated group and 4 in control). Other dead or euthanizedpigs had no gross pathologic signs. One of characterizations of S. suisinfection is that acute infection can be fatal without appreciable grosssigns of pathology. The tracheobronchial lymph node was enlarged. Therewas a trace of fibrin on the mesentery, indicating a mild peritonitis.There was evidence of arthritis in both stifles, in which there was asmall amount of purulent material, and in the left shoulder, where therewas a trace of purulent exudate.

Antibody Response

Immunization of pigs with Sao in combination with Quil A elicitedsignificant humoral IgG responses after primary immunization and abooster injection significantly enhanced the antibody titre (FIG. 23).Furthermore, while both IgG1 and IgG2 subclasses were induced, IgG2titer dominated over IgG1 as measured in the sera 2 weeks after thesecond vaccination (FIG. 24).

Summary and Discussion

The vaccine was shown to be safe since pigs that were vaccinated twicedid not have any adverse reaction. Immunization of pigs with Sao incombination with Quil A elicited significant IgG titres with a dominantIgG2 production, suggesting a predominant Th1-type immune response.Aerosol challenge of pigs resulted in disease with an overall mortalityrate of approximately 58% in controls. The survival of vaccinated pigsafter challenge was significantly better than controls (p<0.05). Somepigs in each group became ill after challenge, and there wassignificantly less disease (lower clinical score) in the vaccinatedpigs. Vaccination had no significant effect on the occurrence of grosspathology post-mortem; however acute streptococcal septicaemia can befatal without appreciable gross signs of pathology. Less S. suisbacteria were recovered from vaccinated pigs than control pigs postmortem (p<0.01).

Example 5 Immunization of Mice and Piglets with Fragment SP1A

Immunization of Mice

Three groups of 10 mice were immunized two times (Day 1 and Day 17) viai.p. with 40 μg of purified SP1A-maltose-binding protein (MBP) fusionprotein, 20 μg of MBP or only PBS, using Freund Incomplete as anadjuvant. The sera were obtained before each immunization or 10 daysafter the second injection, and were 1:5000 diluted for ELISA assay.(See Table 5)

Immunization of Pigs

Three groups of 3 pigs were immunized two times (Day 1 and Day 17) viai.m. with 200 μg of purified SP1A-MBP fusion protein, 100 μg of MBP oronly PBS, using Emulsigen as an adjuvant. The sera were obtained beforeeach immunization or 10 days after the second injection, and were 1:5000diluted for ELISA assay. (See Table 6)

TABLE 1 Distributions of SP1 in S. suis reference strains, isolates ofserotype 2 and other organisms detected by SP1-specific antibody R44 inWestern blots. S. suis serotype S. suis isolate (reference strain)Origin SP1 of serotype 2 Origin SP1  1 (5428) The Netherlands + 89-999Canada + ½ (2651) The Netherlands + 90-1330 Canada +  2 (NCTC 10234) TheNetherlands + 95-8242 Canada +  3 (4961) Denmark + Man 25 Canada +  4(6407) Denmark + Man 50 Canada +  5 (11538) Denmark + Man 63 Canada +  6(2524) Denmark + AAH4 USA +  7 (8074) Denmark + AAH5 USA +  8 (14636)Denmark + AAH6 USA +  9 (22083) Denmark + 1309 USA + 10 (4417) Denmark +88-5955 USA + 11 (12814) Denmark + 95-13626 USA + 12 (8830) Denmark +95-16426 USA + 13 (10581) Denmark − 95-7220 USA + 14 (13730) TheNetherlands + 97-8506 USA + 15 (NCTC 1046) The Netherlands + SX-332USA + 16 (2726) Denmark − JL 590 Mexico + 17 (93A) Canada + 166 France +18 (NT77) Canada + 96-39247 France + 19 (42A) Canada + 96-49808 France +20 (86-5192) USA − 96-53405 France + 21 (14A) Canada + Italie 57 Italy +22 (86-1861) Canada − Italie 68 Italy + 23 (89-2479) Canada + Italie 69Italy − 24 (88-5299A) Canada − Italie 228 Italy + 25 (89-3576-3)Canada + S735^(a) The Netherlands + 26 (89-4109-1) Canada + 27 (89-5259)Canada + 28 (89-590) Canada + 29 (92-1191) Canada + 30 (92-1400)Canada + 31 (92-4172) Canada + 33 (EA1832.92) Canada + Organism StrainSP1 1 2 S. bovis ATCC 9809 − 3 4 S. equisimilis ATCC 9542 − 5 6 S.intestinalis ATCC 43492 − 7 8 S. pyogenes ATCC 14289 − 9 10  S. uberisATCC 6580 − ^(a)Strain used as reference in this work.

TABLE 2 Protection of pigs following challenge with S. suis strain 166Arthritic Bacteremic Surviving Groups (n = 8) pigs pigs pigsEmulsigen-Plus (Control) 6 3 5 Emulsigen-Plus + SP1 4 3 5

TABLE 3 Level of S. suis bacteremia. Groups Significance Sao + Quil-AQuil-A (p) Before challenge ND (12) ND (12) N/A 3 days after challenge1/11 2/12 0.6 Postmortem 1/11 4/9  0.13

TABLE 4 Level of infection postmortem. Groups Brain Lymph node JointMedian bacteriology score Sao + Quil-A  2/11 6/11 2/11 1.0 Quil-A 10/128/12 4/12 4.5

TABLE 5 SP1A-specific IgG response in mouse sera (A450 nm) GroupSP1A-MBP MBP PBS Before Immunization 0.006 0.016 0.0 After 1^(st)Immunization 2.809 0.015 0.005 After 2^(nd) Immunization 3.153 0.0150.004

TABLE 6 SP1A-specific IgG response in pigs (A450 nm) Group SP1A-MBP MBPPBS Before Immunization 0.024 0.018 0.024 After 1^(st) Immunization0.254 0.026 0.015 After 2^(nd) Immunization 0.501 0.047 0.033

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The invention claimed is:
 1. An isolated polypeptide comprising an aminoacid sequence having at least 75% identity to the amino acid sequenceset forth in SEQ ID NO: 1 or in SEQ ID NO:
 4. 2. The isolatedpolypeptide of claim 1, comprising at least one repetitive amino acidsequence consisting of the amino acid sequence Xaa₁ Ser Xaa₃ Xaa₄ Xaa₅Met Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Pro Xaa₁₈Xaa₁₉ Gln Met Xaa₂₂ Asn Lys Glu Xaa₂₆ Xaa₂₇ Xaa₂₈ Xaa₂₉ Xaa₃₀ SEQ ID NO9) wherein Xaa₁ is Val, Thr or Ile; Xaa₃ is Lys or Glu; Xaa₄ is Lys orGlu; Xaa₅ is Ala or Gln; Xaa₇ is Thr or Pro; Xaa₈ is Gly, Ser or Val;Xaa₉ is Lys, VaI, Ile or Asn; Xaa₁₀ is Glu or Val; Xaa₁₁ is Lys or Asn;Xaa₁₂ is Gly, Glu or Asp; Xaa₁₃ is Asn or Met; Xaa₁₄ is Ile, Ala or Val;Xaa₁₅ is Glu or Val; Xaa₁₆ is Pro or Thr; Xaa₁₈ is Glu or Gln; Xaa₁₉ isLys or Glu; Xaa₂₂ is Thr or Ala; Xaa₂₆ is Lys or Asn; Xaa₂₇ is Asp orGlu; Xaa₂₈ is Asn or Lys; Xaa₂₉ is Ile or Val and Xaa₃₀ is Glu or Val.3. The isolated polypeptide of claim 1, comprising the amino acidsequence as set forth in SEQ ID NO
 10. 4. The isolated polypeptide ofclaim 1, comprising an amino acid having at least 85% identity to theamino acid sequence set forth in SEQ ID NO 1 or in SEQ ID NO
 4. 5. Theisolated polypeptide of claim 1, comprising an amino acid sequencehaving at least 95% identity to the amino acid sequence set forth in SEQID NO
 1. 6. The isolated polypeptide of claim 1, comprising an aminoacid sequence having at least 95% identity to the amino acid sequenceset forth in SEQ ID NO
 4. 7. The isolated polypeptide of claim 1,wherein it elicits a protective response to a Streptococcus suis strainchallenge when administered to an animal.
 8. The isolated polypeptide ofclaim 3, comprising at least one repetitive amino acid sequenceconsisting of the amino acid sequence Xaa₁ Ser Xaa₃ Xaa₄ Xaa₅ Met Xaa₇Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Pro Xaa₁₈ Xaa₁₉ GlnMet Xaa₂₂ Asn Lys Glu Xaa₂₆ Xaa₂₇ Xaa₂₈ Xaa₂₉ Xaa₃₀ SEQ ID NO 9) whereinXaa₁ is Val, Thr or Ile; Xaa₃ is Lys or GIu; Xaa₄ is Lys or GIu; Xaa₅ isAla or Gln; Xaa₇ is Thr or Pro; Xaa₈ is Gly, Ser or Val; Xaa₉ is Lys,VaI, Ile or Asn; Xaa₁₀ is Glu or Val; Xaa₁₁ is Lys or Asn; Xaa₁₂ is Gly,Glu or Asp; Xaa₁₃ is Asn or Met; Xaa₁₄ is Ile, Ala or Val; Xaa₁₅ is Gluor Val; Xaa₁₆ is Pro or Thr; Xaa₁₈ is Glu or Gln; Xaa₁₉ is Lys or Glu;Xaa₂₂ is Thr or Ala; Xaa₂₆ is Lys or Asn; Xaa₂₇ is Asp or Glu; Xaa₂₈ isAsn or Lys; Xaa₂₉ is Ile or Val and Xaa₃₀ is Glu or Val.
 9. The isolatedpolypeptide of claim 4, comprising at least one repetitive amino acidsequence consisting of the amino acid sequence Xaa₁ Ser Xaa₃ Xaa₄ Xaa₅Met Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Pro Xaa₁₈Xaa₁₉ Gln Met Xaa₂₂ Asn Lys Glu Xaa₂₆ Xaa₂₇ Xaa₂₈ Xaa₂₉ Xaa₃₀ (SEQ ID NO9) wherein Xaa₁ is Val, Thr or Ile; Xaa₃ is Lys or GIu; Xaa₄ is Lys orGIu; Xaa₅ is Ala or Gln; Xaa₇ is Thr or Pro; Xaa₈ is Gly, Ser or Val;Xaa₉ is Lys, VaI, Ile or Asn; Xaa₁₀ is GIu or Val; Xaa₁₁ is Lys or Asn;Xaa₁₂ is Gly, Glu or Asp; Xaa₁₃ is Asn or Met; Xaa₁₄ is Ile, Ala or Val;Xaa₁₅ is Glu or Val; Xaa₁₆ is Pro or Thr; Xaa₁₈ is Glu or Gln; Xaa₁₉ isLys or Glu; Xaa₂₂ is Thr or Ala; Xaa₂₆ is Lys or Asn; Xaa₂₇ is Asp orGlu; Xaa₂₈ is Asn or Lys; Xaa₂₉ is Ile or Val and Xaa₃₀ is Glu or Val.10. The isolated polypeptide of claim 5, comprising at least onerepetitive amino acid sequence consisting of the amino acid sequenceXaa₁ Ser Xaa₃ Xaa₄ Xaa₅ Met Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄Xaa₁₅ Xaa₁₆ Pro Xaa₁₈ Xaa₁₉ Gln Met Xaa₂₂ Asn Lys Glu Xaa₂₆ Xaa₂₇ Xaa₂₈Xaa₂₉ Xaa₃₀ (SEQ ID NO 9) wherein Xaa₁ is Val, Thr or Ile; Xaa₃ is Lysor GIu; Xaa₄ is Lys or GIu; Xaa₅ is Ala or Gln; Xaa₇ is Thr or Pro; Xaa₈is Gly, Ser or Val; Xaa₉ is Lys, VaI, Ile or Asn; Xaa₁₀ is Glu or Val;Xaa₁₁ is Lys or Asn; Xaa₁₂ is Gly, Glu or Asp; Xaa₁₃ is Asn or Met;Xaa₁₄ is Ile, Ala or Val; Xaa₁₅ is Glu or Val; Xaa₁₆ is Pro or Thr;Xaa₁₈ is Glu or Gln; Xaa₁₉ is Lys or Glu; Xaa₂₂ is Thr or Ala; Xaa₂₆ isLys or Asn; Xaa₂₇ is Asp or Glu; Xaa₂₈ is Asn or Lys; Xaa₂₉ is Ile orVal and Xaa₃₀ is Glu or Val.
 11. The isolated polypeptide of claim 6,comprising at least one repetitive amino acid sequence consisting of theamino acid sequence Xaa₁ Ser Xaa₃ Xaa₄ Xaa₅ Mel Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Pro Xaa₁₈ Xaa₁₉ Gln Met Xaa₂₂ AsnLys Glu Xaa₂₆ Xaa₂₇ Xaa₂₈ Xaa₂₉ Xaa₃₀ (SEQ ID NO 9) wherein Xaa₁ is Val,Thr or Ile; Xaa₃ is Lys or GIu; Xaa₄ is Lys or GIu; Xaa₅ is Ala or Gln;Xaa₇ is Thr or Pro; Xaa₈ is Gly, Ser or Val; Xaa₉ is Lys, VaI, Ile orAsn; Xaa₁₀ is GIu or Val; Xaa₁₁ is Lys or Asn; Xaa₁₂ is Gly, Glu or Asp;Xaa₁₃ is Asn or Met; Xaa₁₄ is Ile, Ala or Val; Xaa₁₅ is Glu or Val;Xaa₁₆ is Pro or Thr; Xaa₁₈ is Glu or Gln; Xaa₁₉ is Lys or Glu; Xaa₂₂ isThr or Ala; Xaa₂₆ is Lys or Asn; Xaa₂₇ is Asp or Glu; Xaa₂₈ is Asn orLys; Xaa₂₉ is Ile or Val and Xaa₃₀ is Glu or Val.
 12. The isolatedpolypeptide of claim 1, comprising an amino acid sequence having 100%identity to the amino acid sequence set forth in SEQ ID NO
 1. 13. Theisolated polypeptide of claim 1, comprising an amino acid sequencehaving 100% identity to the amino acid sequence set forth in SEQ ID NO4.
 14. An isolated polypeptide, comprising an amino acid sequence havingat least 95% identity to the amino acid sequence set forth in SEQ ID NO2.
 15. The isolated polypeptide of claim 14, comprising an amino acidsequence having 100% identity to the amino acid sequence set forth inSEQ ID NO
 2. 16. An isolated polypeptide, comprising an amino acidsequence having at least 95% identity to the amino acid sequence setforth in SEQ ID NO
 3. 17. The isolated polypeptide of claim 16,comprising an amino acid sequence having 100% identity to the amino acidsequence set forth in SEQ ID NO
 3. 18. A composition comprising anacceptable carrier and at least one of the following elements: apolypeptide as defined in any one of claims 1, 2, 3, 4, 5, 6, 7, and 8to
 17. 19. A composition for eliciting an immune response toStreptococcus or treating Streptococcus suis-associated diseases orinfection caused by S. suis, comprising an acceptable carrier and atleast one of the following elements: a polypeptide as defined in any oneof claims 1, 2, 3, 4, 5, 6, 7, and 8 to
 17. 20. The composition of claim19, further comprising an adjuvant.
 21. A method of screening for apolypeptide having immunogenic potential for preventing a Streptococcussuis-associated disease, comprising: (a) administering to an animal, atest polypeptide comprising at least 15 contiguous amino acids of theamino acid sequence of SEQ ID NO: 4; and (b) processing a biologicalsample from said animal to detect the presence or absence in said sampleof an antibody having binding specificity to the polypeptide, whereinthe presence of said antibody is indicative of the polypeptide havingimmunogenic potential for preventing the Streptococcus suis-associateddisease.
 22. The method of claim 21, wherein the test polypeptidecomprises at least 25 contiguous amino acids of the amino acid sequenceof SEQ ID NO:
 4. 23. The method of claim 21, wherein the testpolypeptide comprises at least 35 contiguous amino acids of the aminoacid sequence of SEQ ID NO:
 4. 24. The method of claim 21, wherein thetest polypeptide comprises the amino acid sequence of SEQ ID NO:
 9. 25.The method of claim 24, wherein the amino acid sequence of SEQ ID NO: 9is a first repetitive amino acid sequence of a plurality of saidrepetitive amino acid sequence.
 26. The method of claim 21, wherein saidpolypeptide is a recombinant polypeptide.
 27. The method of claim 21,wherein said animal is a pig.
 28. A method for preventing aStreptococcus suis-associated disease in an animal, comprisingadministering to the animal a polypeptide comprising at least 15contiguous amino acids of the amino acid sequence of SEQ ID NO:
 4. 29.The method of claim 28, said animal being a pig.
 30. A microbialculture, comprising cells having a recombinant DNA molecule encoding apolypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence set forth in SEQ ID NO:
 4. 31. Amethod for producing a polypeptide comprising an amino acid sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 4,comprising cultivating a microbial culture comprising cells having arecombinant DNA molecule encoding said polypeptide under conditionsconducive for production of said polypeptide, and recovering saidpolypeptide.
 32. A method for preventing a Streptococcus suis-associateddisease in an animal, comprising administering to the animal apolypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO:
 4. 33. The method ofclaim 28, wherein the polypeptide comprises at least 25 contiguous aminoacids of the amino acid sequence of SEQ ID NO:
 4. 34. The method ofclaim 28, wherein the polypeptide comprises at least 35 contiguous aminoacids of the amino acid sequence of in SEQ ID NO: 4.